Genetically modified organisms are increasingly used to produce human consumables such as fuels (e.g., ginkgo, LS9, SOLAZYME, Chromatin), commodity chemicals (e.g., GENENCOR, GENOMATICA, VERDEZYNE), and therapeutics (e.g., AMBRX, AMYRIS). They are also used in agriculture (e.g., GOLDEN RICE, ROUNDUP READY crops, FROSTBAN), bioremediation (e.g., oil spills), and healthcare (e.g., Crone's disease and oral inflammation). Although bio-containment strategies, such as engineered auxotrophy, induced lethality and gene flow prevention have been developed, selective pressure from leaky containment mechanisms can lead to bio-containment escape.
Genetically modified organisms provide unique opportunities to broaden the functional repertoire accessible to biotechnology. By reassigning the genetic code, organisms can produce proteins incorporating nonstandard amino acids (NSAAs) with properties not found in nature, resist viral infection due to mistranslated viral transcripts, and maintain genetic isolation from naturally coded organisms in their surroundings (F. J. Isaacs et al. (2011) Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement. Science 333:348). However, such organisms require unprecedented safety mechanisms to ensure their containment. Current best practices confer dependence on supplemented metabolites like diaminopimelic acid (DAP) (R. Curtiss (1978) Biological containment and cloning vector transmissibility. Journal of Infectious Diseases 137:668); J. Santander, W. Xin, Z. Yang, R. Curtiss (2010) The Aspartate-Semialdehyde Dehydrogenase of Edwardsiella ictaluri and Its Use as Balanced-Lethal System in Fish Vaccinology. PLoS One 5:e15944), an essential component of the cell wall, for survival. Since metabolites like DAP are natural products found in the environment, this strategy is insufficient to ensure containment of genetically modified organisms. Mutations that suppress NSAA dependence, acting either on the recoded genes or, more likely, on extent tRNAs (G. Eggertsson, D. Söll (1988) Transfer ribonucleic acid-mediated suppression of termination codons in Escherichia coli. Microbiological Reviews 52:354), will compromise any safety features that rely on genomic recoding.
Auxotrophies for natural metabolites like DAP or Thy are known in the art (R. Curtiss, Supra; J. Santander, Supra), but they lack sufficient safeguards. This is due to the fact that the metabolites can be found in nature, safety tests often focused only on the mammalian gut, and conjugation tests often considered only conjugation out from a genetically modified organism rather than into it, and did not address transduction by viruses and or phages.
Kill switches controlling genetically modified organism survival are known in the art (M. C. Ronchel, J. L. Ramos (2001) Dual system to reinforce biological containment of recombinant bacteria designed for rhizoremediation. Applied and Environmental Microbiology 67:2649), but these too lack sufficient safeguards. These genetically modified organisms have the potential for escape via failure of the killing mechanism and/or via broken circuitry (e.g., inactivation of repressors, activators, or other expression regulation mechanisms involved in the kill switch, loss of expression of a killing mechanism, constitutive activity of a survival mechanism and the like).
Methods of placing NSAAs at surface positions or near the 5′ translation start site are also known, although these methods are also lacking. These methods incorporate natural amino acids at surface or terminal positions and are far less likely to disrupt folding and function, leaving clear routes to natural suppression (i.e., a natural tRNA mutates so that is can incorporate a natural amino acid at the desired position of the NSAA). Although escape can potentially be mitigated by placing NSAAs at very many surface and terminal positions, this complicates the approach relative to a much fewer number of high-impact core mutations and doesn't protect against suppressor mutations in which a natural amino acid is translated at the reassigned codon.
Horizontal and vertical gene transfer methods are also known. Agriculture and bioremediation use genetically modified organisms in the environment. However, methods are needed to prevent escape of herbicide/antibiotic resistant strains and to prevent cross-pollination and/or DNA transfer between genetically modified organisms and organic crops or other natural organisms.
Partial methods for reassigning the genetic code in order to efficiently incorporate NSAAs during translation are known (T. Mukai et al. (2010) Codon reassignment in the Escherichia coli genetic code. Nucleic Acids Res. 38:8188; D. Johnson et al., Rfl knockout allows ribosomal incorporation of unnatural amino acids at multiple sites. Nat Chem Biol. 7:779), but these methods are lacking. For example, partial recoding methods are not scalable/generalizable to other codons, because all genes would have to be duplicated using the target codon. Additionally, UAG function cannot be abolished because these methods do not remove all instances of UAG throughout the genome. Accordingly, such methods only swap codon function. Furthermore, these methods result in a strong selection pressure for natural suppressors, which leads to an unstable genetic code.
The engineering of organisms that can only grow safely in well-defined, restrictive environments has been a long-standing goal that dates back to the Asilomar Conference from 1975 and has yet to be achieved. Accordingly, recombinant cells, recombinant organisms (and methods of making them) that avoid unintended survival are needed.